The present invention relates to synthetic promoter and a synthetic gene which confers broad-spectrum disease resistance to Xanthomonands in plants. The present invention also relates to transgenic plants containing the synthetic gene and plants derived by crossing plants with such transgenic plants. More specifically, the synthetic promoter is a synthetic Xa10 promoter and the synthetic gene is a synthetic Xa10 gene which contains the synthetic Xa10 promoter. The resistance is resistance to bacterial blight and the plants are rice plants.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.
Gram-negative phytopathogenic bacteria employ a type III secretion system (TTSS) to translocate effector proteins into plant cells where they modulate host cell functions for the benefit of the invasion process (Alfano and Collmer, 2004; Kay and Bonas, 2009). Members of the large AvrBs3 effector family are unique kind of type III effectors produced by pathovars of Xanthomonas and Ralstonia solanacearum (Bonas et al., 1989; Yang and White, 2004; Heuer et al., 2007). AvrBs3-like effectors, also recently referred to as transcription activator-like (TAL) effectors (Yang et al., 2006; Bogdanove et al., 2010), have in common an N-terminus required for type III secretion and a C-terminus containing nuclear localization signals (NLS) and an acidic activation domain (AAD). TAL effectors differ in the middle, a region of typically 33-35 amino acid (aa) long, near-perfect repeats that ends in a 20 aa long truncated repeat. The two hypervariable amino acids at position 12 and 13 of repeats, also termed as repeat-variable di-residue (RVD) (Moscow and Bogdanove, 2009), contribute to repeat polymorphism, whereas the number and order of repeats with polymorphic RVDs in a TAL effector determine the specific activity (Herbers et al., 1992; Yang et al., 2005).
Individual TAL effectors activate transcription of specific host susceptibility (S) genes for promoting disease development (Yang et al., 2006; Kay et al., 2007; Sugio et al., 2007; Antony et al., 2010). In order to counteract the disease-promoting strategies, plants have evolved mechanisms that exploit the transcription inducing ability of the TAL effectors (Gu et al., 2005; Romer et al., 2007). Thus, a subset of TAL effectors functions as avirulence effectors and activates the transcription of disease R genes. TAL effectors bind specific DNA sequences in the promoters of S or R genes (Kay et al., 2007; Romer et al., 2007; Romer et al., 2009; Antony et al., 2010). Each RVD from the central repeats of TAL effectors specifically recognizes a nucleotide in the target DNA element with a conserved T at the 5′ end (Boch and Bonas, 2010; Bogdanove et al., 2010).
Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice (Nino-Liu et al., 2006). Individual strains of X. oryzae pv. oryzae harbor 11 to 19 TAL effectors (White et al., 2009). TAL effectors from X. oryzae pv. oryzae target to rice genes for either susceptibility (Yang et al., 2006; Sugio et al., 2007; Chen et al., 2010) or resistance to bacterial infection (Gu et al., 2005). TAL effector PthXo1 from X. oryzae pv. oryzae strain PXO99A targets Os8N3/Xa13/OsSWEET11 in rice (Yang et al., 2006; Chen et al., 2010). The recessive allele of Xa13 (xa13) is unresponsive to PthXo1, and plants with xa13 are resistant to strains of the pathogen that rely solely on the PthXo1 as the essential effector for virulence (Yang et al., 2006). The xa13-mediated resistance to rice bacterial blight can be defeated by induction of the S gene Os-11N3, another member of the N3 gene family, by strains of the pathogen utilizing the TAL effectors AvrXa7 and PthXo3 (Antony et al., 2010).
PthXo6 and PthXo7 are two other TAL effectors from PXO99A and target respectively to two transcription factor genes, OsTFX1 and OsTFIIA γ1, in rice (Sugio et al., 2007). OsTFX1 encodes a bZIP transcription factor whereas the gene product of OsTFIIA γ1 is the small subunit of the transcription factor HA (Sugio et al., 2007). The induction of OsTFIIA γ1 located on chromosome 1 by PthXo7 may reflect the adaptation of PXO99A to the resistance mediated by xa5 (Iyer and McCouch, 2004), an allele of OsTFIIA γ5 encoding a second form of the TFIIA small subunit on chromosome 5 of rice (Sugio et al., 2007). The DNA target sequences of PthXo1, EBEPthXo1 (Effector Binding Element for PthXo1), has been identified in the promoter of Os8N3 (Antony et al., 2010), whereas the DNA target sequences of PthXo6 and PthXo7 remain to be identified or verified in the promoters of OsTFX1 and OsTFIIA γ1, respectively, although the putative target sequences were predicted (Boch et al., 2009).
Three TAL effectors, AvrXa7 and AvrXal10 from PXO86 (Hopkins et al., 1992) and AvrXa27 from PXO99A (Gu et al., 2005), activate disease resistance when rice plants carried the cognate R genes Xa7, Xa10 or Xa27. So far, only the Xa27 gene has been isolated and published (Gu et al., 2005). Xa27 is induced by AvrXa27 and the gene can provide non-specific resistance to X. oryzae pv. oryzae if the AvrXa27-inducible promoter is replaced with a stress-inducible promoter from the rice PR1 gene (Gu et al., 2005; Tian and Yin, 2009). The full induction of Xa27 by AvrXa27 requires OsTFIIA γ5, the gene product of Xa5 on chromosome 5 (Gu et al., 2009). A 16- to 18-bp DNA cis-element, designated as UPTAvrXa27 (UPregulated by TAL effector AvrXa27 or EBEAvrXa27), was identified in the promoter of Xa27 to be specifically induced by AvrXa27 (Boch et al., 2009; Romer et al., 2009).
Xa10 confers narrow-spectrum race-specific resistance to a few Philippine races of X. oryzae pv. oryzae (Yoshimura et al., 1995). The R gene was introgressed from rice cultivar Cas 209 into susceptible rice variety IR24 (Mew, 1982; Yoshimura et al., 1983). Xa10 was finely mapped to a genetic region of 0.28 cM between proximal marker M491 and distal marker M419 on the long arm of chromosome 11 and co-segregated with markers S723 and M604 (Gu et al., 2008). The Xa10 gene was recently cloned by map-based cloning and genetic transformation approaches (International Published Application No. WO 2012/033462). A functional target sequence of AvrXa10, EBEAvrXa10 was identified in the promoter of Xa10 (International Published Application No. WO 2012/033462). The Xa10 gene product, XA10, is functional in both monocots and dicots by inducing hypersensitive response (HR)-like cell death (unpublished).
The resistance specificity of TAL effector-dependent R gene to bacterial blight is determined by the R gene promoter rather than the R gene products (Gu et al., 2005). Meanwhile, the spectrum of TAL effector-dependent R genes to bacterial blight varies greatly, which is depended on the availability of the avirulence TAL effectors in the X. oryzae pv. oryzae strains (Gu et al., 2004; Gu et al., 2008). Romer et al (2009) demonstrated that multiple functionally distinct DNA elements targeted by separate TAL effectors retain their function and specificity when combined into one promoter. It is desired to generate broad-spectrum resistance to bacterial blight in rice.